a) Field of the Invention
This invention relates generally to cloned cells, embryos and animals, and to methods of producing them via nuclear transfer and combinations of nuclear transfer and ovum transfer. In particular, this invention relates to the cloning of avians.
b) Description of Related Art
A major aim of biotechnologists is to produce offspring from cell populations that can be maintained in culture and to obtain stable desirable phenotypes that transmit the required traits through the germ line. For nuclear transfer, the availability of cultured cell lines facilitates cell cycle synchronization of the donor nucleus and allows optimization of cell cycle co-ordination in the reconstituted embryo. In order to obtain stable desirable phenotypes, nuclear transfer from cultured cell populations provides a promising alternative route to genetic modification. Until recently, genetic modification in farm animals was limited to pronuclear injection wherein the required gene is injected into the pronucleus of a zygote. Although pronuclear injection has been applied in several species including mice, rabbits, pigs, sheep, goats and cattle, there are a number of disadvantages with this technique. Integration of the gene does not always occur during the first cell cycle, resulting in the production of mosaic embryos. In addition, integration occurs at random within the genome, resulting in variable expression of the gene product. Thus, the production of an animal having the required phenotype capable of germ line transmission may require the generation of several transgenic lines, as well as extensive breeding of founder animals. In comparison, the production of animals by nuclear transfer from cells that can be maintained in culture offers a number of advantages over pronuclear injection. In nuclear transfer, the cells to be used as nuclear donor cells may be sexed, optionally genetically modified, and selected in culture before their use. The resultant animal is produced from a single nucleus and mosaics can therefore be avoided. The genetic modification is easily transmitted to the offspring. In addition, all the cells in the animal are likely to contain the transgene and expression can be obtained in the tissues of interest.
The ability to produce live offspring by nuclear transfer from cultured somatic cells also provides a route for the precise genetic manipulation of animal species. Such modifications include the addition or “knock-in” of genes, and the removal or inactivation or “knock-out” of genes or their control sequences (Polejaeva et al., Theriogenology, 53(1):117–26, 2000). Gene targeting techniques also promise the generation of transgenic animals in which specific genes coding for endogenous proteins have been replaced by human genes coding for exogenous human proteins. In 1993, Yom and Bremmel suggested that genes coding for major proteins in cow's milk could be replaced by human counterparts. Cows modified in this fashion would produce milk containing human milk proteins, which may be more nutritious for human infants and more suitable for infant formula manufacture (Yom, H. C. and Bremmel, R. D., American Journal of Clinical Nutrition, 1993, 58 (Supplement) 306S). Methods for producing exogenous proteins in the milk of pigs, sheep, goats and cows have been reported.
Wilmut and Campbell (GB 2 331 751 B) have reported the production of reconstituted animal embryos by transferring the nucleus of a diploid donor cell into a suitable recipient cell. The donor cells are quiescent, i.e. not actively proliferating, and are characterized as being in the G0 or G1 phase. The recipient cells include oocytes at metaphase I or II, zygotes, and two-cell embryos, preferably enucleate. Enucleation is accomplished by splitting, aspiration, or irradiation. In those mechanical techniques predicated upon visualization of the nucleus, the use of a DNA-specific fluorochrome is required.
Nuclear transfer (NT) involves the transfer of the complete diploid genetic material (the DNA contained in a nucleus) from a donor cell into an enucleated recipient cell, such as a fertilized (zygote) or unfertilized (oocyte) cell. The technique involves several steps. The donor cells are first grown under special conditions in culture, increasing the number of cells by several orders of magnitude. The nuclei of the donor cells are then transferred to oocytes or zygotes, resulting in a reconstructed embryo. Activation (initiation of development) is usually artificially, most often chemically, induced. The embryos are then transplanted into female animals and allowed to develop to term. In some species (mice, cattle and sheep) the reconstructed embryos may be grown in culture until the blastocyst stage before transfer to a recipient.
The reconstruction of mammalian embryos by transfer of a blastomere nucleus to an enucleated oocyte or zygote has been reported to produce genetically identical individuals (Eyestone et al., J. Reprod. Fertil. Suppl,. 1999, 54:489–97). Although the number of offspring that can be produced from a single embryo is limited both by the number of blastomeres (embryos at the 32–64 cell stage are the most widely used in farm animal species) and the efficiency of the nuclear transfer procedure, the ability to produce live offspring by nuclear transfer from cells that can be propagated and maintained in culture offers many advantages. This includes the production of identical offspring over an extended period (since cultured cells can be frozen and stored indefinitely) and the ability to genetically modify and to select populations of cells of specific genotypes or phenotypes before embryo reconstruction. This objective has been achieved with the production of lambs using nuclei from cultured cells established from embryonic, fetal and adult material.
Two types of recipient cells are commonly used for nuclear transfer: oocytes arrested at the metaphase of the second meiotic division (MII) and pronuclear zygotes. In mice, enucleated, two-cell stage blastomeres have been used as recipients. In farm species, development does not always occur when pronuclear zygotes are used, except when pronuclei are exchanged between zygotes, therefore, MII-arrested oocytes have often been the recipient of choice. Oocytes arrested at MII do not contain a nucleus but a metaphase plate, where the chromosomes are arranged on the meiotic spindle. The MII chromosomes or metaphase plate are not easily visible under the light microscope in mammalian oocytes. However, visualization of the MII chromosomes or metaphase plate has been achieved with UV light using DAPI (4′,6′-diamidino-2-phenylindole, hydrochloride) or Hoescht 33342 (bis-benzimide) staining. After enucleation and introduction of the donor genetic material, the reconstructed embryo must be cultured to a stage at which it can be transferred to a recipient animal, generally the morula or blastocyte stage. This can be done in vitro or in vivo (Eyestone et al., supra). Double nuclear transfer has also been reported, in which an activated, previously transferred nucleus is removed from the host unfertilized egg and transferred a second time into an enucleated fertilized embryo. (Polejaeva et al., Nature, 407:505-9, 2000).
Although gene targeting techniques in combination with nuclear transfer hold tremendous promise with respect to nutritional and medical applications, current approaches suffer from several limitations, including long generation times between founder and production transgenic herds, and extensive husbandry and veterinary costs. It is therefore desirable to employ a system where the use of cultured somatic cells for nuclear transfer is more efficiently employed. One system that holds great potential is the avian reproductive system.
The avian reproductive system, including that of the chicken, is well described. The production of an egg begins with formation of the large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. Upon ovulation or release of the yolk from the ovary, it passes into the infundibulum of the oviduct where it is fertilized if sperm are present. It then moves into the magnum of the oviduct which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum from which they are deposited onto the avian embryo and yolk.
Attempts at nuclear transfer in avians have remained difficult to realize. One significant challenge is the inaccessibility of the early avian egg. It is well known in the art that all forms of genetic manipulation that require visualization of the avian early embryo have been hindered by the inability to properly visualize the target structures. In nuclear transfer, after enucleation, the genetic material from the donor cell (nuclear donor) must be introduced into the enucleated oocyte. In order to produce an enucleated recipient cytoplast, it is essential to visualize the metaphase II plate or pronuclei that reside about 25 μm beneath the egg's vitelline membrane within the germinal disk. Yet, the large size and optical density of the yolk have made visualization of the avian early embryo and its structures difficult to achieve.
The hen oviduct offers outstanding potential as a protein bioreactor because of high levels of protein production, proper folding and post-translation modification of the target protein, and ease of product recovery. As a result, efforts have been made to create transgenic chickens expressing exogenous proteins in the oviduct by means of microinjection of DNA (PCT Publication WO 97/47739). Bosselman et al. describe a method for introducing a replication-defective retroviral vector into a pluripotent stem cell of an unincubated chick embryo, and further describe chimeric chickens, whose cells express an exogenous vector nucleic acid sequence. However, the percentage of G1 transgenic offspring (progeny from vector-positive male G0 birds) was low and varied between 1% and approximately 8% (U.S. Pat. No. 5,162,215). Generally, DNA injection into avian eggs has so far lead to poor and unstable transgene integration (Sang and Perry, Mol. Reprod. Dev., 1:98–106, 1989, and Naito, et al., Mol. Reprod. Dev. 37:167–71, 1994). In addition, the use of viral vectors poses a number of limitations, including limited transgene size and potential viral infection of the offspring. The production of transgenic chickens by means of DNA microinjection (supra) is both inefficient and time-consuming. In fact, a key limitation of using any animal as a bioreactor is the time required (approximately 10 months for chickens, 2–3 years for ungulates) to introduce the desired transgene into the animal's genome.
The hen also offers a unique system for efficient direct transgenesis of the magnum gland, but initial attempts have yielded poor results. Plasmid DNAs carrying transgenes have been introduced directly into the magnum of mature hens by electroporation (Ochiai et al., Poultry Science, 77:299–302, 1998). Due to the large size of the oviduct of mature hens, the transient persistence of the plasmid DNAs in the cells, and the inefficiency of organ electroporation, only very low levels of protein were detected in the oviduct tissue of sacrificed hens and no expressed protein was reported as being detected in the egg. Other attempts have involved the transfection of magnum cells with expression cassettes after excision of the cells from the bird and preparation of an oviduct cell culture (Sanders et al., Endocrinology, 116:398–405, 1985; Sanders et al., Biochemistry, 27:6550–6557, 1988; Schweers et al., Journal of Biological Chemistry, 265:7590–7595, 1990; Otten et al., Molecular Endocrinology, 2:143–147, 1988; Schweers et al., Journal of Biological Chemistry, 266:10490–10497, 1991).
Ovum transfer, the transfer of a donor ovum to the oviduct of a recipient hen, provides another means for genetic manipulation in avians. Tanaka et al. produced chicks by in vitro fertilization (IVF) by returning the fertilized ovum into the oviduct of a recipient hen to complete the egg and shell formation. This experimental approach suggests a useful model for production of transgenic avians (Tanaka et al., Journal of Reproduction and Fertility, 100:447–449, 1994).
Another major challenge is the culture of the reconstructed egg. It is essential to hatch the reconstructed zygote following micromanipulation. One solution was proposed by Perry et al. (U.S. Pat. No. 5,011,780). Perry et al. use ex ovo culture to remove an embryo and yolk from a donor hen and incubate the embryo and yolk in a series of separate culture systems until hatch. Yet, this procedure is laborious and inefficient with low numbers of “test tube chickens” hatching.
It is an object of this invention to provide an improved method for visualization of the nuclear structures in a recipient cell to facilitate the process of enucleation and subsequent nuclear transfer.
It is also an object of this invention to provide an improved method for ablation of the nucleus in a recipient cell to facilitate subsequent nuclear transfer.
It is a particular object of the instant invention to provide methods that overcome the technical hurdles relating to the cloning of avians, such as inaccessibility of the avian egg and difficulty of culturing the reconstructed zygote. A new and useful method for successful nuclear transfer in avians in order to produce cloned birds would satisfy a present need in the art. Avians cloned in this manner may be genetically modified. The resulting expression and deposition of exogenous proteins in eggs, suitable for commercial use, would provide immediate benefits to the public.